U.S. patent application number 11/448778 was filed with the patent office on 2007-12-13 for nanoparticles comprising lanthanide chelates.
This patent application is currently assigned to WALLAC OY. Invention is credited to Harri Hakala, Jari Hovinen, Veli-Matti Mukkala.
Application Number | 20070286810 11/448778 |
Document ID | / |
Family ID | 38822235 |
Filed Date | 2007-12-13 |
United States Patent
Application |
20070286810 |
Kind Code |
A1 |
Hovinen; Jari ; et
al. |
December 13, 2007 |
Nanoparticles comprising lanthanide chelates
Abstract
The invention concerns a method for preparing a particle
comprising a lanthanide chelate, comprising polymerizing a
lanthanide chelate derivative with one or more monomers, wherein
the lanthanide chelate derivative has the formula M-L-Y (I),
wherein Y is a lanthanide chelate; L is a linker and M is a
polymerizable moiety. The invention concerns also the novel
lanthanide chelate derivative M-L-Y (I). Further, the invention
concerns a novel particle comprising a lanthanide chelate
covalently bound to an organic polymer or copolymer via a
linker.
Inventors: |
Hovinen; Jari; (Raisio,
FI) ; Mukkala; Veli-Matti; (Kaarina, FI) ;
Hakala; Harri; (Turku, FI) |
Correspondence
Address: |
BUCHANAN, INGERSOLL & ROONEY PC
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
WALLAC OY
Turku
FI
|
Family ID: |
38822235 |
Appl. No.: |
11/448778 |
Filed: |
June 8, 2006 |
Current U.S.
Class: |
424/9.322 ;
525/245; 534/16 |
Current CPC
Class: |
C07D 409/04 20130101;
A61K 49/0052 20130101; A61K 49/0002 20130101; C08F 230/04 20130101;
A61K 49/0019 20130101; C07D 401/06 20130101 |
Class at
Publication: |
424/9.322 ;
534/16; 525/245 |
International
Class: |
A61K 49/10 20060101
A61K049/10; C07F 5/00 20060101 C07F005/00; C08F 277/00 20060101
C08F277/00 |
Claims
1. A method for preparing a particle comprising a lanthanide
chelate, comprising polymerizing a lanthanide chelate derivative
with one or more monomers, wherein the lanthanide chelate
derivative has the formula M-L-Y (I), wherein Y is a lanthanide
chelate; L is a linker and M is a polymerizable moiety.
2. The method according to claim 1 wherein the polymerizable moiety
M is selected from pendent vinyl groups, acrylate groups,
methacrylate groups, ethacrylate groups, 2-phenylacrylate groups,
vinylketone groups, acrylamide groups, methacrylamide groups,
itaconate groups and styrene groups.
3. The method according to claim 2 wherein M is methacrylamide or
acrylamide.
4. The method according to claim 1 wherein the lanthanide chelate
derivative is a compound of formula (I) and the one or more
monomers are selected from styrene, vinyl alcohol, acrylic acid,
metacrylic acid monomers or esters or amides thereof.
5. The method according to claim 1 wherein the lanthanide chelate
derivative is a compound of formula (I) and the one or more
monomers are styrene monomers.
6. The method according to claim 1 wherein the lanthanide chelate
is a compound of formula (I) selected from the group of:
##STR00007## wherein R is independently furyl, thiophenyl, or
trialkoxyphenyl; n is independently 1 or 2; and L is a linker; Ln
is europium, terbium, samarium, dysprosium or gadolinium; and M is
a polymerizable moiety.
7. The method according to claim 1 wherein the linker -L- is formed
from one to ten moieties, each moiety being selected from the group
consisting of phenylene, alkyl containing 1-12 carbon atoms,
ethynediyl(--C.ident.C--), ethylenediyl (--C.dbd.C--); ether
(--O--), thioether (--S--), amide (--CO--NH-- and --NH--CO-- and
--CO--NR' and --NR'--CO--), carbonyl(--CO--), ester (--COO-- and
--OOC--), disulfide (--SS--), diaza, (--N.dbd.N--), thiourea
(--NH--CS--NH--) or a tertiary amine (--NR'--), where R' represents
an alkyl containing less than 5 carbon atoms.
8. The method according to claim 1, wherein the particle has a
diameter of less than 500 nm.
9. The method according to claim 1, wherein the polymerizing is
performed in the presence of a cross-linking agent.
10. The method according to claim 9, wherein the cross-linking
agent is divinylbenzene.
11. A compound of the formula M-L-Y (I), wherein Y is a lanthanide
chelate; L is a linker and M is a polymerizable moiety.
12. The compound according to claim 11, wherein M is selected from
pendent vinyl groups, acrylate groups, methacrylate groups,
ethacrylate groups, 2-phenylacrylate groups, vinylketone groups,
acrylamide groups, methacrylamide groups, itaconate groups and
styrene groups.
13. The compound according to claim 12, wherein M is methacrylamide
or acrylamide.
14. The compound according to claim 11, selected from the group of:
##STR00008## wherein R is independently furyl, thiophenyl, or
trialkoxyphenyl; n is independently 1 or 2; and L is a linker; Ln
is europium, terbium, samarium, dysprosium or gadolinium; and M is
a polymerizable moiety.
15. The compound according to claim 11 wherein the linker -L- is
formed from one to ten moieties, each moiety being selected from
the group consisting of phenylene, alkyl containing 1-12 carbon
atoms, ethynediyl(--C.ident.C--), ethylenediyl(--C.dbd.C--); ether
(--O--), thioether (--S--), amide (--CO--NH-- and --NH--CO-- and
--CO--NR' and --NR'--CO--), carbonyl(--CO--), ester (--COO-- and
--OOC--), disulfide (--SS--), diaza, (--N.dbd.N--), thiourea
(--NH--CS--NH--) or a tertiary amine (--NR'--), where R' represents
an alkyl containing less than 5 carbon atoms.
16. A particle comprising a compound of formula (II) Z-L-Y (II)
wherein, Z is an organic polymer or copolymer L is a linker; and Y
is a lanthanide chelate, wherein the lanthanide chelate Y is
covalently bound to Z via the linker L.
17. The particle according to claim 16 where the diameter of the
particle is less than 500 nm.
18. The particle according to claim 16 wherein the linker -L- is
formed from one to ten moieties, each moiety being selected from
the group consisting of phenylene, alkyl containing 1-12 carbon
atoms, ethynediyl(--C.ident.C--), ethylenediyl(--C.dbd.C--); ether
(--O--), thioether (--S--), amide (--CO--NH-- and --NH--CO-- and
--CO--NR' and --NR'--CO--), carbonyl(--CO--), ester (--COO-- and
--OOC--), disulfide (--SS--), diaza, (--N.dbd.N--), thiourea
(--NH--CS--NH--) or a tertiary amine (--NR'--), where R' represents
an alkyl containing less than 5 carbon atoms.
19. The particle according to claim 16 wherein L-Y of compound (II)
is selected from ##STR00009## wherein R is independently furyl,
thiophenyl, or trialkoxyphenyl; n is independently 1 or 2; L is a
linker; and Ln is europium, terbium, samarium, dysprosium or
gadolinium.
20. The particle according to claim 16 wherein the organic polymer
or copolymer is constructed from monomers of vinyl, acrylate,
methacrylate, ethacrylate, 2-phenylacrylate, vinylketone, vinyl
alcohol, acrylamide, methacrylamide, itaconate or styrene.
21. The particle according to claim 16 constructed with a
cross-linking agent.
22. The particle according to claim 21 wherein the cross-linking
agent is divinylbenzene.
Description
FIELD OF THE INVENTION
[0001] This invention relates to particles comprising luminescent
lanthanide chelates and to a method for their preparation.
BACKGROUND OF THE INVENTION
[0002] The high specific activity and very low background signal
has made time-resolved fluorescence (TR-F) based on lanthanide(III)
chelates a succesful detection technology for a variety of
analytes. Indeed, lanthanide(III) chelates have been used in in
vitro diagnostics over two decades.
[0003] The commercially available DELFIA.RTM. assay method uses a
non-luminescent europium(III) chelate for detecting a target
molecule. The usual steps of a DELFIA.RTM. assay include capturing
of the target molecule to a surface using a target-specific
antibody associated with a lanthanide chelate, such as a europium
(III) chelate, washing away of contaminating materials, and signal
enhancement. For the signal enhancement, europium(III) ion is
dissociated from the non-luminescent chelate by lowering pH to 3.2
and the luminescence is enhanced with a mixture of .beta.-diketone
(4,4,4-trifluoro-1-(2-napthyl)-butane-1,3-dione), detergent (Triton
X) and chelator (trioctyl phosphine oxide, TOPO). The new chelate
formed has a very high luminescence, giving detection sensitivity
ca 5010.sup.-15 M.
[0004] Luminescent lanthanide(III) chelates with increased
stability have been developed more recently. These chelates consist
of a ligand with a reactive group for covalent conjugation to
bioactive molecules, an aromatic structure, which absorbs the
excitation energy and transfers it to the lanthanide ion and
additional chelating groups such as carboxylic acid moieties and
amines.
[0005] Further improvement to lanthanide(III) chelate materials and
methods would allow for development of more sensitive assay
methods, and increased ability to detect target molecules.
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention provides a method preparing a
particle comprising a lanthanide chelate, wherein a lanthanide
chelate derivative of the formula M-L-Y (I), wherein Y is a
lanthanide chelate; L is a linker and M is a polymerizable moiety,
is allowed to polymerize with one or more monomers.
[0007] According to another aspect, the invention provides a
lanthanide chelate derivative of the formula M-L-Y (I), wherein Y
is a lanthanide chelate; L is a linker and M is a polymerizable
moiety.
[0008] According to a third aspect, the invention provides a
particle based on a compound of formula Z-L-Y (II)
[0009] wherein,
[0010] Z is an organic polymer or copolymer
[0011] L is a linker; and
[0012] Y is a lanthanide chelate,
[0013] wherein the lanthanide chelate Y is covalently bound to Z
via the linker L.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The technology described herein relates to particles linked
covalently to luminescent lanthanide(III) chelates. The particles
are prepared by polymerizing lanthanide(III) chelate derivatives in
the presence of monomers. Because the resultant particles are
covalently bound to the lanthanide(III) chelates, the signal
obtained generally does not decrease as the function of time due to
leaking upon storage. Because the lanthanide(III) chelates are
luminescent, no additives that promote luminescence, such as
phosphine oxides, are needed in the polymerization matrix. In
addition, because the beads can be made using organic polymers or
copolymers, such as polystyrene, they can be stable under basic
conditions.
[0015] It has been shown previously that lanthanide (III) chelate
detection sensitivity can be enhanced by incorporating chelates
into particles. Beads containing lanthanide(III) chelates have most
commonly been prepared simply by swelling the chelates into the
polymer, as is described, for example, in Cummins, C. M., Koivunen,
M. E., Stephanian, A., Gee, S. J., Hammock, B. D., Kennedy, I. M.,
2006, Biocencors and Bioelectronics, 21, 1077. In addition, silica
based nanobeads have been described by Hai, X., Tan, M., Wang, G.,
Ye, Z., Yuan, J., Matsumoto, K., 2004, Anal. Sci., 20, 245 and Sun,
X., Wuest, M., Kovacs, Z., Sherry, A. D., Motekaitis, R., Wang, Z.,
Martell, A. E., Welch, M. J., Anderson, C. J., 2003, J. Biol.
Inorg. Chem., 8, 217. Particles produced using nanodispersions of
particle sizes in the middle or lower nanometer range (50-500 nm)
are described, for example, in Horn, D., Rieger, J., 2001, Angew.
Chem. Int. Ed. Engl., 40, 4330.
[0016] The invention provides a method for preparing particles
comprising a lanthanide chelate. The method involves polymerizing a
lanthanide chelate derivative with one or more monomers, wherein
the lanthanide chelate derivative has the formula M-L-Y (I),
wherein Y is a lanthanide chelate; L is a linker and M is a
polymerizable moiety.
[0017] In an embodiment, the polymerizable moiety M in the
lanthanide chelate derivative of formula (I) is a pendent vinyl
group, acrylate group, methacrylate group, ethacrylate group,
2-phenylacrylate group, vinylketone group, acrylamide group,
methacrylamide group, itaconate group or a styrene group. Specific
examples include methacrylamide or acrylamide.
[0018] The compound of formula (I) can be allowed to polymerize
with one or more organic monomers. Examples of organic monomers
include styrene, vinyl alcohol, acrylic acid, metacrylic acid and
esters and amides derived thereof. A specific example monomer is
styrene.
[0019] The lanthanide chelate can be a luminescent lanthanide
chelate. In an embodiment, the lanthanide chelate derivative is
typically made of a chromophoric moiety comprising one or more
aromatic units, and a chelating part. Exemplary compounds include
those of shown below:
##STR00001##
[0020] wherein
[0021] R is independently furyl, thiophenyl, or
trialkoxyphenyl;
[0022] n is independently 1 or 2; and
[0023] L is a linker; and
[0024] Ln is europium, terbium, samarium, dysprosium or gadolinium;
and
[0025] M is a polymerizable moiety.
[0026] The linker -L- is formed from one to ten moieties, each
moiety being selected from the group consisting of phenylene, alkyl
containing 1-12 carbon atoms, ethynediyl(--C.ident.C--),
ethylenediyl(--C.dbd.C--); ether (--O--), thioether (--S--), amide
(--CO--NH-- and --NH--CO-- and --CO--NR' and --NR'--CO--),
carbonyl(--CO--), ester (--COO-- and --OOC--), disulfide (--SS--),
diaza, (--N.dbd.N--), thiourea (--NH--CS--NH--) or a tertiary amine
(--NR'--), where R' represents an alkyl containing less than 5
carbon atoms.
[0027] The polymerization can be performed in the presence of a
cross-linking agent such as divinylbenzene.
[0028] The size of the particle is generally below 500 nm, such as
below 300 nm. In an embodiment, the diameter of the particle is in
the range 10 . . . 150 nm, such as 90 . . . 120 nm.
[0029] The organic polymer or copolymer of the particle can be
constructed, for example, of from monomers of vinyl, acrylate,
methacrylate, ethacrylate, 2-phenylacrylate, vinylketone, vinyl
alcohol, acrylamide, methacrylamide, itaconate or styrene.
EXAMPLES
[0030] The technology described herein is further elucidated by the
following non-restricting examples. The structures and synthetic
routes employed in the experimental part are depicted in Schemes 1
and 2. Experimental details are given in Examples 1-9. Scheme 3
describes a schematic preparation of a polymerizable terbium
chelate. Properties of the nanospheres prepared are collected in
Table 1.
Procedures
[0031] Adsorption column chromatography was performed on columns
packed with silica gel 60 (Merck). All dry solvents were from Merck
and they were used as received. NMR spectra were recorded on a
Brucker 250 on a Jeol LA 400 spectrometer operating at 250.13 and
399.7 MHz for .sup.1H, respectively. The signal of TMS was used as
an internal reference. Coupling constants are given in Hertz.
ESI-TOF mass spectra were recorded on an Applied Biosystems Mariner
instrument. Luminescence measurements were measured with a
PerkinElmer LS-5 luminescence spectrometer. IR and UV-spectra
spectra were recorded on a PerkinElmer Spectrum One and Shimatzu
2400 instruments, respectively. Particle size analyses were
performed on a Coulter LS-230 instrument, and are based on volume
statistics. Photophysical properties of the nanoparticles prepared
were measured as disclosed in Latva, M., Takalo, H., Mukkala,
V.-M., Matachescu, C., Rodriques-Ubis, J. C., Kankare, J., 1997, J.
Lumin., 35, 149 but the chelate concentration measurements were
based on the weight of dry beads.
Example 1
The Synthesis of N-(3-acetylphenyl)methacrylamide (1)
[0032] 3-Aminoacetophenone (20.9 g, 0.15 mol) was dissolved in dry
pyridine (60 mL) on an ice-water bath. Methacryloyl chloride (26.1
mL, 0.24 mol) was added dropwise during 1/2 h, and the mixture was
stirred for an additional 1/2 h. The stirring was continued for 3 h
at RT. All volatiles were removed in vacuo. The residue was
dissolved in dichloromethane (150 mL), washed with 0.5 M HCl (2100
mL) and water (2100 mL) and dried (Na.sub.2SO.sub.4). Purification
was performed on silica gel. The column was first eluted with
CH.sub.2Cl.sub.2 to elute fast migrating impurities, and then with
5% (v/v) methanolic dichloromethane to elute the product. Yield was
18.1 g (59%). .sup.1H NMR (CDCl.sub.3): 8.10 (1H, m); 8.00 (1H,
br); 7.95 (1H, m); 7.43 (1H, m); 7.23 (1H, m); 5.85 (1H, s); 5.51
(1H, s); 2.61 (3H, s); 2.08 (3H, s). ESI-TOF MS: required for
C.sub.12H.sub.14NO.sub.2.sup.+ 204.10 (M+H),.sup.+ found
204.08.
Example 2
The Synthesis of
4,4,4-Trifluoro-1-[3-(methacrylamido)phenyl]-1,3-butanedione
(2)
[0033] To a strirred solution of compound 1 (11.9 g, 58.55 mmol) in
dry THF (100 mL) was added portionwise sodium hydride (3.52 g, 88
mmol; 60% dispersion in oil). After 5 min, ethyl trifluoroacetate
(13.9 mL, 0.18 mol) was added, and the mixture was strirred for an
additional 1 h before being concentrated in vacuo. The residue was
suspended in ethyl acetate (220 mL) and acidified with 10% aqueous
H.sub.2SO.sub.4 (80 mL), and washed with water. The organic layer
was separated and dried over Na.sub.2SO.sub.4. Purification on
silica gel (eluent, petroleum ether, bp. 40-60.degree. C./ethyl
acetate, 1:1, v/v) yielded 8.71 g (50%) of the title compound.
.sup.1H NMR (CDCl.sub.3): 8.14 (1H, s); 7.87 (1H, d, J 7.5); 7.74
(1H, br s); 7.74 (1H, d, J 7.8); 7.48 (1H, t, J 8.0); 6.58 (1H, s);
5.85 (1H, s); 5.54 (1H, s); 2.09 (3H, s). .lamda..sub.max(EtOH)/nm:
208, 250, 326. ESI-TOF MS: required for
C.sub.13H.sub.13F.sub.3NNaO.sub.3.sup.+ 322.07 (M+Na),.sup.+ found
322.03.
Example 3
The Synthesis of the Europium Chelate (3)
[0034] Compound 2 (9.8 g, 32.7 mmol) was dissolved in the mixture
of abs. ethanol (100 mL) and piperidine (3.2 mL), and the mixture
was warmed to 45.degree. C. Europium chloride hexahydrate (2.40 g,
6.54 mmol, predissolved in 20 mL of water) was added dropwise. The
mixture was allowed to cool to RT, and water (100 mL) was added
dropwise. The precipitation formed was collected by filtration,
washed with water, and dried in vacuo. Yield was 7.9 g.
.lamda..sub.max(H.sub.2O+1% DMF, v/v)/nm: 326 (.epsilon. 50552).
.nu./.sub.max(KBr) cm.sup.-1: 3440, 1663, 1620, 1586, 1534, 1489,
1301, 1187, 1138, 780, 580. Ex.sub.max 614 nm; Em.sub.max 353 nm
(tris-saline buffer, pH 7.75).
Example 4
The Synthesis of
tetra-(tert-butyl)-2,2',2'',2'''-{[6-(tert-butyloxycarbonylamino)hexylimi-
no]bis(methylene)bis(4-bromopyridine-6,2-diyl)bis
methylenenitrilo)}tetrakis(acetate) (5)
[0035] 4-Bromo-6-bromomethyl-2-pyridylmethylenenitrilobis(acetic
acid) di(tert-butyl ester) (4, 8.50 g, 16.7 mmol) and
6-tert-butoxycarbamoylhexane-1,6-diamine (1.80 g, 8.4 mmol) were
dissolved in dry acetonitrile (60 mL). K.sub.2CO.sub.3 (9.2 g, 66.8
mmol) was added, and the mixture was heated overnight at 50.degree.
C. The precipitation formed was removed by filtration, and the
filtrate was concentrated. Purification on silica gel (eluent:
petroleum ether, bp 40-60.degree. C.: ethyl acetate, from 10:1 to
5:2, v/v) yielded 5.9 g (65%) of compound 5. .sup.1H NMR
(CDCl.sub.3): .delta. 7.73 (2H, d, J 1.9); 7.58 (2H, d, J 1.9);
4.00 (4H, s); 3.74 (4H, s); 3.47 (8H, s); 3.08 (2H, q, J 5.5); 2.51
(2H, t, J 7.2); 1.46 (36H, s); 1.44 (9H, s); 1.53-1.42 (4H, m);
1.33-1.22 (4H, m). .nu./.sub.max(film) cm.sup.-1: 3401 (N--H); 1734
(C.dbd.O); 1565 (arom. C--C). .lamda..sub.max(EtOH)/nm 268. ESI-TOF
MS for C.sub.49H.sub.78Br.sub.2N.sub.6O.sub.10 (M+2H).sup.2+:
calcd, 536.22; found, 536.18.
Example 5
The Synthesis of
tetra-(tert-butyl)-2,2',2'',2'''-{[6-(tert-butyloxycarbonylamino)hexylimi-
no]bis(methylene)bis(4-(thiophen-2-yl)pyridine-6,2-diyl)bis
methylenenitrilo)}tetrakis(acetate) (6)
[0036] Compound 5 (2.85 g, 2.66 mmol) and
2-(tributylstannyl)-thiophene (1.86 mL, 5.86 mmol) were dissolved
in dry DMF (25 mL) and deaerated with argon. (Ph.sub.3P).sub.4Pd
(0.215 g, 0.22 mmol) was added, and the mixture was stirred at
90.degree. C. for 6 h in dark. The mixture was cooled to room
temperature and concentrated in vacuo. Purification was performed
on silica gel (eluent: petroleum ether, bp 40-60.degree. C.: ethyl
acetate: triethylamine, from 5:1:1 to 5:3:1, v/v/v). Yield was 2.2
g (76%). .sup.1H NMR (CDCl.sub.3): .delta. 7.76 (2H, s); 7.70 (2H,
s); 7.55 (2H, d J 3.1); 7.36 (2H, d, J 4.9); 7.09 (2H, m); 4.05
(4H, s); 3.82 (4H, s); 3.50 (8H, s); 3.03 (2H, m); 2.60 (2H, m);
1.49-1.43 (4H, m); 1.45 (36H, s); 1.42 (9H, s); 1.39-1.32 (4H, m).
.nu..sub.max (film)/cm.sup.-1 1730 (C.dbd.O).
.lamda..sub.max(EtOH)/nm 293. ESI-TOF-MS for
C.sub.57H.sub.85N.sub.6O.sub.10S.sub.2(M+2H).sup.2+: calcd, 539.29;
obsd, 539.23.
Example 6
The Synthesis of
2,2',2'',2'''-{(6-Aminohex-1-yl-imino)bis(methylene)bis[4-(thiophen-2-yl)-
pyridine-6,2-diyl)]bis(methylenenitrilo)}tetrakis(acetic acid)
(7)
[0037] Compound 6 (2.16 g, 2.00 mmol) was dissolved in
trifluoroacetic acid (25 mL), and the mixture was stirred for 2 h
at room temperature before being concentrated. The residue was
triturated with diethyl ether. The precipitation formed was
filtered, washed with diethyl ether and dried in vacuo. Yield was
quantitative. .sup.1H NMR (DMSO-d.sub.6): 7.90 (2H, s); 7.78 (2H,
d, J 4.9); 7.73 (2H, d, J 3.4); 7.71 (2H, s); 7.25 (2H, dd, J 3.4
and 4.9); 3.95 (4H, s); 3.30 (8H, s); 3.22 (2H, m); 2.74 (2H, m);
1.78 (2H, m); 1.50 (2H, m); 1.33-1.23 (4H, m);
.nu..sub.max(KBr)/cm.sup.-1 1735, 1675, 1609 (C.dbd.O); 1559 (arom.
C--C). .lamda..sub.max(EtOH)/nm 300.
Example 7
The Synthesis of
2,2',2'',2'''-{(6-aminohex-1-yl-imino)bis(methylene)bis[4-(thiophen-2-yl)-
pyridine-6,2-diyl)]bis(methylenenitrilo)}tetrakis(acetic acid)
europium(III) (8)
[0038] Compound 7 (1.9 g) was dissolved in water (30 mL), and pH of
the solution was adjusted to 6.5 with solid NaHCO.sub.3. Europium
chloride hexahydrate (0.81 g, 2.2 mmol; predissolved in 30 mL of
water) was added dropwice keepimg pH at ca 6. The mixture was
stirred for 1.5 h at RT. pH was rised to 8.5 with aq. NaOH. The
precipitation was removed by centrifugation. The clear solution was
collected and concentrated in vacuo. It was used for the next step
without further purification. .nu..sub.max(KBr)/cm.sup.-1 1684,
1638, 1615 (C.dbd.O); 1552 (arom. C--C). .lamda..sub.max(EtOH)/nm
312. ESI-TOF-MS for
C.sub.36H.sub.40EuN.sub.6O.sub.8S.sub.2.sup.-(M-H).sup.-: calcd,
901.16; obsd, 901.16.
Example 8
The Synthesis of
2,2',2'',2'''-{(6-methacroylamidohex-1-yl-imino)bis(methylene)bis[4-(thio-
phen-2-yl)pyridine-6,2-diyl)]bis(methylenenitrilo)}tetrakis(acetic
acid) europium(III) (9)
[0039] Compound 8 (2.6 g, 2.8 mmol) was dissolved in the mixture of
water (20 mL), THF (40 mL) and DIPEA (1.7 mL). Methacroyl chloride
(0.42 g, 4.0 mmol) was added, and the mixture was stirred for 5 min
at RT before being concentrated in vacuo. The residue was suspended
in chloform (30 mL). The precipitation formed was removed by
filtration. The filtrate was concentrated to give the title
compound. ESI-TOF-MS for
C.sub.40H.sub.44EuN.sub.6O.sub.9S.sub.2.sup.- (M-H).sup.-: calcd,
969.18; obsd, 969.18. The partition coefficient of compound 9
between H.sub.2O and CHCl.sub.3, was ca 1:1.
Example 9
Polymerization. A Typical Procedure
[0040] A mixture of styrene (1.83 mL), acrylic acid (234 mm.sup.3),
hexadecane (94 mg), divinyl benzene (0.12 g) and compound 3 (0.512
g, 0.358 mmol; 20% of the dry weight; predissolved in 2.0 mL of
chloroform) and TOPO (0.208 g, 0.534 mmol) were dissolved in water
(40.0 mL) containing sodium dodecyl sulfate (0.09 g) and
sodiumborate decahydrate (0.017 g). The resulting suspension was
deaerated with argon and homogenized using ultrasound (1 min, 215
W). The resulting emulsion was transferred into a reactor, and it
was stirred mechanically (140 rpm) at 60.degree. C. for 20 min
under argon (pH 3). The polymerization was initiated by addition of
potassium persulphate (0.05 g, predissolved in 3.00 mL of degassed
water). The reaction was allowed to proceed for 5 h. The mixture
was allowed to cool to RT and purified by dialysis. The following
beads were prepared:
[0041] 10% (w/w) chelate 3 [bead A]
[0042] 20% (w/w) chelate 3 [bead B]
[0043] 20% (w/w) chelate 3+1.5 equiv. TOPO, [bead C]
[0044] 27% (w/w) chelate 9 [bead D]
[0045] where % w/w is the of the weight of chelate from the dry
weight of the polymerization mixture.
TABLE-US-00001 TABLE 1 Properties of the nanospheres prepared. Bead
size (nm) chelate (w/w).sup.a relative fluorescence.sup.b A 101 3
(10%) 0.097.sup.c (0.19).sup.d B 115 3 (20%) 0.210 (0.39) C 111 3
(20%) + TOPO 1.52 (1.40) D 93 9 (27%) 0.42 (0.42) .sup.apercentage
of dry weight of the chelate in the polymerization mixture;
.sup.bcompared to DELFIA enhancement solution; .sup.cmeasured in
Tris-saline buffer, pH 7.75; .sup.dTris-saline buffer, pH 7.75 +
TOPO + Triton-X.
[0046] It will be appreciated that the methods described herein can
be incorporated in the form of a variety of embodiments, only a few
of which are disclosed herein. It will be apparent for the expert
skilled in the field that other embodiments exist and do not depart
from the spirit of the invention. Thus, the described embodiments
are illustrative and should not be construed as restrictive.
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